The present invention relates to multiple array systems for integrating arrays of biomolecules, including biological, chemical and biochemical elements.
There is a need to rapidly assay compounds for their effects on various biological processes. Nearly all biological activity is regulated by the interactions of proteins in cells. Proteins are the catalysts, motion transducers, and signal mediators of cells. They control cell division, cell growth, cell differentiation, cell death, and mediate the responses of cells to their environments. Enzymologists have long sought better substrates, better inhibitors and better catalysts for enzymatic reactions. To understand cellular processes, we therefore need to monitor the activity of proteins, and to determine the networks of interactions of proteins within cells.
In the past, tools available to biologists only allowed the study of one interaction at a time because there were no analytical tools that would allow large numbers of protein interactions to be monitored simultaneously. Thus, a system that would allow parallel analyses of protein interactions would be of immense value and would speed the progress of biological discovery.
In addition, there is a need to rapidly assay or screen compounds for potential drug candidates. Drug discovery is a long, multiple step process involving the identification of specific disease targets, development of assays based on a specific target, validation of the assays, and optimization and automation of the assay to achieve screening of a large number of candidates. After high throughput screening of compound libraries using various assays, hit validation and hit compound optimization procedures are employed. Performing a screen on many thousands of compounds thus requires parallel processing of many compounds and assay component reagents. In addition, to find lead compounds for drug discovery programs, large numbers of compounds are often screened for their activity as enzyme inhibitors or receptor agonists/antagonists. Large libraries of compounds are needed for such screening. As a result of developments in this field, it is now possible to simultaneously produce combinatorial libraries containing hundreds of thousands of small molecules for screening. With such libraries on hand there is an ever increasing need to rapidly screen the thousands of these new potential drug candidates.
One common approach to drug discovery involves presenting macromolecules implicated in causing a disease (disease targets) in bioassays in which potential drug candidates are tested for therapeutic activity. Such molecules could be receptors, enzymes, transcription factors, co-factors, DNA, RNA, growth promoters, cell-death inducers, or non-enzymatic proteins and peptides. Another approach involves presenting whole cells or organisms that are representative of the causative agent of the disease. Such agents include bacteria and tumor cell lines. Thus, there is a need to be able to screen the effects of various drug candidates on assorted cells and cell lines.
The conventional methods for assessing the effects of various agents or physiological activities on biological materials utilize standard microtiter plates. See for example, U.S. Pat. No. 6,083,763. Unfortunately microtiter plates do not allow for expansion into multiple parameter assays. For example, assessment of the effect of a physiological agent, such as a drug, on a population of cells or tissue grown in culture conventionally provides information relating to the effect of the agent on the cell or tissue population only at specified points in time. In addition, current assessment techniques generally only provide information relating to a single or a small number of parameters. For example, candidate agents are systematically tested for cytotoxicity, which may be determined as a function of concentration. A population of cells is treated, and at one or several time points following treatment, cell survival is measured. Thus, cytotoxicity assays generally do not provide any information relating to the cause(s) or time course of cell death but merely show whether cells die or survive. To elucidate the mechanism of the interaction/activity of agents, an assay capable of simultaneously monitoring several parameters is required.
In addition, therapeutic agents are frequently evaluated based on their physiological effects on a particular metabolic function. An agent is administered to a population of cells or a tissue sample, and the metabolic function of interest is assayed to assess the effect of the agent. This type of assay provides useful information, but it does not provide information relating to the mechanism of action, the effect on other metabolic functions, the time course of the physiological effect, general cell or tissue health, and the like.
Despite the great value that screening libraries of molecules has for identifying useful pharmaceutical compounds and improving the properties of a lead compound, the difficulties of screening, and especially the lack of xe2x80x9cfunctionalxe2x80x9d screening methods, of these libraries has limited the impact that these methods have had in drug discovery and development. Thus, there remains a need for an assay system that allows a simultaneous screen for multiple target-ligand interactions in drug discovery and in the development of lead compounds. There exists a strong need for a high throughput multilevel assay system to test potential drug candidates and to obtain biologically and clinically relevant information. This need is not limited to drug discovery but also concerns diagnostic and clinical diagnosis arenas as well.
The relationship between structure and function of molecules is a fundamental issue in the study of biological systems. Structure-function relationships are important for understanding biological phenomena such as enzyme function, cellular communication, and cellular control and feedback mechanisms to name a few. Understanding how various molecules interact with each other, such as protein-receptor interactions for example, often provides the first step in understanding biomolecule function.
Modern pharmaceutical drug discovery often relies on the study of structure-function relationships. Much contemporary drug discovery involves discovering novel ligands with desirable patterns of specificity for biologically important receptors. Thus, the length of time necessary to bring new drugs to market could be greatly reduced by assay systems that allow rapid screening of structure-function relationships of large numbers of ligands.
Within the general drug discovery strategies, several sub-strategies have been developed. One spatially-addressable strategy that has emerged involves the generation of peptide libraries on immobilized pins that fit the dimensions of a standard 96 well micro-titer plate. See PCT Patent Publication Nos. 91/17271 and 91/19818, each of which is incorporated herein by reference. This method has been used to identify peptides that mimic discontinuous epitopes as described in Geysen et al., xe2x80x9cScreening Chemically Synthesized Peptide Libraries for Biologically Relevant Molecules,xe2x80x9d Bioorg Med Chem. Lett. 3: 397-404 (1993), and to generate benzodiazepine libraries as described in U.S. Pat. No. 5,288,514 and Bunin et al., xe2x80x9cThe Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-Benzodiazepine Library,xe2x80x9d Proc. Natl. Acad Sci. 91:4708-4712 (1994). The structures of the individual library members can be determined from the pin location in the micro titer plate and the sequence of reaction steps (called a xe2x80x9csynthesis histogramxe2x80x9d) performed during the synthesis.
In addition to the above-mentioned methods used in drug discovery, several trends are fueling interest in the application of cell-based assays for drug discovery. Cell-based screens offer the potential to shorten the time between target validation and lead drug discovery because these assays can be miniaturized to increase screening throughput and reduce costs. Information about target biology is encouraging development of assays for specific, often subtle effects on target function. For example, cell based assays have been used to screen for modulators of ion channel kinetics, allosteric regulators of receptor agonist efficacy and protein interactions. Unfortunately, these assays are often difficult to format using traditional ligand displacement or biochemical methods because the binding sites of modulators may be unknown.
Advances in biology, chemistry and instrumentation have provided user-friendly tools for the development of optical indicators of cell function. Many of these tools allow direct detection of target function in living cells. Most of these tools employ either fluorescent or luminescent reporter molecules and allow cell-based assays for most targets, including receptors, ion channels and intracellular enzymes. Examples of these tools include adsorbance assays, fluorescence intensity assays, fluorescence resonance energy transfer (xe2x80x9cFRETxe2x80x9d) assays, fluorescence distribution assays, fluorescence polarization, and luminescence assays. These novel optical assays promise to accelerate the use of living cells in screens for drug discovery.
Common therapeutic targets for high throughput screening (xe2x80x9cHTSxe2x80x9d) are enzymes, cell surface receptors, nuclear receptors, ion channels, signal transduction proteins, cell surface glycoproteins and proteoglycans. Compounds that interact with these targets are usually identified using in vitro biochemical assays.
Cell-based assays using engineered mammalian cell lines provide the most biologically relevant information because the ligand interaction occurs in the biological environment of the target. This provides opportunities to simultaneously monitor secondary cellular events. Thus, because of the numerous novel cell-based assays that are currently available there is a need for a multiple level array system that can be used in conjunction with these cell-based assays. The present invention addresses this need and specifically provides for high density arrays that can be used in cell based assays.
These assays and other conventional assays, as well as HTS, such as DNA analysis, gene expression profiling, mapping for single nucleotide polymorphisms (SNP""s), and enzyme linked immunosorbent assay (ELISA) and others rely on arraying of biomolecules.
In a recent development, the techniques of photolithography, chemistry and biology have been combined to array large collections of biocompounds on the surface of a substrate. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. 90/15070 and 92/10092, each of which is incorporated herein by reference.
xe2x80x9cGenechipxe2x80x9d technology as well as photolithography to generate patterns of target oligonucleotides (See U.S. Pat. No. 5,599,695) have revolutionized the ways assays are performed. Robotic spotting systems have been developed to xe2x80x9cprintxe2x80x9d arrays of nucleic acids and other materials on surfaces for assay development. Unfortunately, both photolithography and robotic array systems require expensive equipment. In addition, processing conditions required in photolithography are often incompatible with many biochemical and biological materials. Robotic spotting systems work well when used with homogeneous fluids, but they do not work efficiently when attempting to pattern cells directly. Although, silkscreen printing and ink-jet printing have recently shown much promise in the generation of biological arrays, they suffer from their inability to generate patterns having fine resolutions. Obtaining sub-100 xcexcm resolution is difficult with these techniques.
Despite these difficulties, protein arrays provide a promising tool that allows biological researchers to perform high throughput assays. Various concepts have been proposed for protein arrays. The most common is composed of arrays of monoclonal antibodies that bind to specific proteins in a way similar to the way that arrays of cDNA capture mRNA. See Arenkov, P., et al., Analytical Biochemsitry, 2000, 278, pp. 128-131. Other approaches include the use of arrays of chemicals which bind proteins. Arrays such as these have been used to isolate proteins, but no array system useful for monitoring interactions between proteins yet exists.
Thus, there is a need for a multilevel array system that can utilize and take advantage of the recent advances involving cell-based assays, lithography, and protein arrays. There is a need for a multiple parameter assay which can perform repeated, accurate assay screens at a very small volume. A new system in which multiple targets and ligands can be identified as new pharmacological, diagnostic, experimental, or other useful agents would fill this need as well as an assay system that has arrays upon arrays to allow for rapid screening of multiple targets, such as cells or proteins, against multiple agents, such as drug-candidate compounds. The present invention provides a multilevel array assay system that can be used in conjunction with standard microtiter plates, such as 6-well, 12-well, 24-well, 96-well, 384-well, or 1536-well microtiter plates, microtiter plate readers and microtiter plate robotic systems. The present invention provides a novel multilevel array system that can be used in HTS, in the study of protein-protein interaction, cell based assays, and other known biological assays. In addition, the present invention provides a multiple array that allows for micro patterning of cells using soft lithography methods, i.e. patterning of cells in an area of about 100 microns in diameter.
The present invention provides multiple level micro arrays as well as a method of preparing the arrays. The arrays can be composed of proteins, nucleic acids, cells, antibodies, enzymes, glycoproteins, proteoglycans, and other biological materials, as well as chemical or biochemical substances. The invention is especially useful in drug discovery and clinical diagnostic applications. Bioarrays can provide large amounts of data about biological systems, such as their disease state or the effect of a drug. These arrays can be integrated into high throughput screening methods that are commonly used in drug discovery, for example, robotic systems. Additionally, the arrays can be designed to take advantage of systems developed for current assay formats, such as detection systems and robotic systems and the like which are designed to handle 6-well, 12-well, 24-well, 96-well, 384-well, 1536-well plates, or even 9,600-microwell plates, for example. The present invention is not limited to the presently used microtiter plate configurations but provides for any configuration necessary to take advantage of today""s industry standards as well as provides the flexibility to design for novel configurations.
The present invention provides a multiple level array system in which a substrate is patterned with a biomolecule, such as a protein or cell, using soft lithography techniques and elastomeric membranes. The multiple levels are achieved by either patterning multiple levels of biomolecules and/or utilizing multiple elastomeric membranes.
A first layer of an elastomeric membrane is used to pattern biomolecules onto a substrate. The membrane has through holes through which the biomolecules are patterned. A second membrane is placed on top of the first membrane, or alternatively, the first membrane is removed and the second is placed directly on the substrate. The second membrane has through holes that define reservoirs that encompass the microwells of the first membrane (or the patterned elements on the substrate).
The ability to pattern proteins in a multilevel array systems offers a vast improvement over the prior art method of assaying a particular protein by adding it to a standard microwell plate. When proteins are simply placed on a surface of a microwell plate, they adsorb non-specifically and denature and thus, loose their activity. The present invention overcomes this problem by arraying proteins on a substrate, preferably a SAM, which allows proteins to maintain their natural configuration and activity.
The present invention also provides for micro arrays of patterned cells in a particular spatial pattern to be later used in various cell based assays.